Correction: Aiphanol, a native compound, suppresses angiogenesis via dual-targeting VEGFR2 and COX2

1 Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Biochemistry and Molecular Biology, Peking University Cancer Hospital & Institute, Beijing 100142, China. 2 Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China. # Current address: Key Laboratory of Molecular Pathology, The Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou, 450008, China.


Evaluation of cellular intake of Aiphanol in cells by immunofluorescence staining
HUVECs or MC38 cells were incubated with FAM-labeled Aiphanol (15 µM) for 6 h. For live cell analysis, the cells were counterstained with 5 µM Hoechst 33342 for 10 min. For fixed cell analysis, the cells were fixed with 4% paraformaldehyde for 20 min, followed by permeabilization with 0.1% Triton X-100 for 5 min and blocked with 5% goat serum for 1h at room temperature. Anti-β-tubulin was applied to the cells overnight at 4°C. After washing with PBST, the cells were probed with Alexa Fluor 594-conjugated secondary antibody and counterstained with 4,6-diamidino-2-phenylindole (DAPI). Images were captured under the Leica TCS-SP2 confocal microscopy (Heidelberg, Germany).

Tube formation assay
In vitro angiogenesis was performed as previous reported. 2 Briefly, the 96-well plate pre-coated with 50 μL growth factor reduced Matrigel was incubated at 37°C for 1 h to solidify as base. Cells were resuspended in serum-free medium containing 0.1% BSA. Each well with polymerized Matrigel was added with 100 μL cell suspension containing 15,000 cells with or without 20 ng/mL of growth factors and indicated chemicals. After 6 h, the tubes were photographed under the microscope and quantified with by the Angiogenesis Analyzer plugin in Image J.

Cell growth assay
The cells were seeded in 96-well plates at a density of 4,000 cells/well, then treated with vehicle or indicated concentrations of SGR or Aiphanol for 24 h. CCK8 reagent from Dojindo (Kumamoto, Japan) was added to wells for 1 h incubation. The amount of formazan salt was determined by measuring the optical density at 450 nm using a Bio-Rad microplate reader. Alternatively, cell confluence rates were detected every 24 h by the CloneSelect Imager from Molecular Devices.

Colony formation assay
For the plate colony formation assay, 1,000 MC38 cells were seeded in 6-well plates and cultured for 12 d.
Colonies were fixed in cold methanol, stained with 0.1% crystal violet, and counted under the microscope. For the soft-agar colony formation assay, 6-well plates were pre-coated with RPMI 1640 medium containing 0.6% agarose as base. After solidification, 1,000 MC38 cells resuspended in RPMI 1640 medium containing 0.35% agarose were seeded. Plates were incubated for 14 d and colonies were counted under microscope.

Apoptosis assay
After treatment with 30 µM Aiphanol for 24 h, HUVECs were harvested and stained with AnnexinV-FITC/PI Apoptosis Detection Kit from Dojindo according to the manufacturer's protocol. Apoptotic cells were measured by flow cytometry (BD Biosciences, USA).

Cell-cycle analysis
After treatment with 30 µM Aiphanol for 24 h, HUVECs were harvested by trypsinization and fixed in 75% ethanol overnight at 4°C. The cells were stained with PI mixture and analyzed by flow cytometry with ModFit LT 3.0 (Verity Software House, USA).

Wound Healing assay
HUVECs were plated in 6-well plates until the confluence reached 90%. A straight wound line was drawn across the attached cell layer. After washing with PBS twice to remove remaining FBS and floating cells, the cells were cultured in serum-free medium and exposed to SGR or Aiphanol. Pictures of the marked gaps were taken after 24 h. The wound gaps were digitally quantified using Image Pro Plus software.

Transwell migration and invasion assay
The migrated HUVECs suspension (40,000 cells in 200 μL) or invaded cells suspension (50,000 cells in 500 μL) was treated with SGR or Aiphanol and seeded into the upper well of transwell inserts (Corning, USA) for 24 h. The lower well was added with EBM-2 medium containing 2% FBS as chemotactic attractors. The cells that have penetrated to bottom side of the transwell membranes were fixed with methanol and stained with 0.1% crystal violet. 5-6 random fields were captured under microscope and the numbers of migrated or invaded cells were counted.

Western Blot
The cells were homogenized in RIPA buffer containing 50 mM Tris-HCl (pH 7.5), 20 mM NaF, 1 mM EDTA, 1 mM Na 3 VO 4 , 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM Dithiothreitol, and 1 x protease inhibitor cocktail from Roche. Protein concentration was quantified by the BCA kit (Thermo Fisher Scientific, USA). Cell lysates (30 μg per sample) were subjected to SDS-PAGE and transferred onto nitrocellulose membranes. After blocking with 5% milk in 0.1% Tween 20/TBS (TBST) for 1 h at room temperature, the nitrocellulose membranes were probed with indicated antibodies for 12 h at 4°C. The protein bands were visualized with Enhanced Chemiluminescence Detection Kit-HRP (Biological Industrials, Israel).

Enzyme linked immunosorbent assay (ELISA)
Levels of VEGF in the conditioned medium or MC38 tumor tissues were measured by VEGF DuoSet from R&D System (Shanghai, China). Levels of PGE2 in the conditioned medium or plasma of mice were analyzed by PGE2 detection kit from Cloud-Clone Corp (Wuhan, China) according to the manufacturer's instructions.
Optical densities at 450 nm were measured with a Bio-Rad microplate reader.

Cell-ELISA
HUVECs were plated in 96-well plates and exposed to Aiphanol (30 μM) for 0.5 to 24 h. After fixation with 2.5% glutaraldehyde and permeabilization with 0.5% TritonX-100, anti-phosphoserine/threonine or anti-phosphotyrosine antibody was added to the wells and incubated for 2 h. After washing with TBST, the plates were incubated with HRP-conjugated anti-mouse-IgG for 1 h. After washing for three times, plates were incubated with 3,3',5,5'-Tetramethylbenzidine (TMB) solution (Cloud-Clone Corp) and stop solution (1N H 2 SO 4 ). Optical densities at 450 nm were quantified using a Bio-Rad microplate reader and represented as relative levels of phospho-proteins.

COX enzymatic activity assay
The COX Fluorescent Screening Assay Kit from Cayman Chemical (Ann Arbor, USA) was utilized according to the manufacturer's instructions to examine the effect of Aiphanol on the activities of COX-1/COX-2 isozymes. The final product resorufin with high fluorescent was analyzed with an excitation wavelength of 530-540 nm and an emission wavelength of 585-595 nm.

Kinase profiler TM assay
The kinase profiler TM assay was performed by Eurofins Cerep SA (Celle L'Evescault, France). Aiphanol (30 μM) was co-incubated with different kinases in the reaction buffer containing 8 mM MOPS (pH 7.0), 0.2 mM EDTA, 0.1% -mercaptoethanol, 10 mM Magnesium Acetate and γ-32 P-ATP. The reaction was initiated by the addition of the Mg/ATP mix. After incubation for 40 min at room temperature, the reaction was stopped by the addition of phosphoric acid. Reaction mix (10 µL) was then spotted onto a P30 filtermat for scintillation counting. The results were calculated using the following formula: (Mean of Sample Counts -Mean of Blank Counts)/Mean of Control Counts.

Molecular docking
Crystal structure of the human COX2 protein (PDB ID: 5IKV) and its selective inhibitor, flufenamic acid, was retrieved from the protein databank. Crystal structure of the human VEGFR2 protein (PDB ID: 4ASE) and its selective inhibitor, Tivozanib, was retrieved as above. The Protein Preparation Wizard Panel was used to modify the three-dimensional (3D) model of COX2 or VEGFR2, including dehydrating, hydrogenating, repairing the missing residue, optimizing the structure, and minimizing the energy (OPLS2005 force field, 0.30 Å RMSD). The box size was 20 Å × 20 Å × 20 Å. 2D format of Aiphanol was dealt with LigPrep module for energy minimization to output the corresponding 3D structure. The sophisticated mode of Glide module was used for molecular docking, that is, the receptors and ligands were docked with each other through geometric and energy matching. The binding patterns of the COX2-Aiphanol and VEGFR2-Aiphanol were plotted in 2D and 3D upon the output.

Binding kinetics analysis with Surface plasma resonance (SPR)
The SPR experiment was performed with a Biacore T200 on CM5 sensor chips from GE Healthcare (Seattle, USA). After activation of CM5 sensor chip, VEGFR2 was diluted to 25 μg/mL in NaAc (pH 5.0) and immobilized at the flow velocity of approximately 10 L/min for 30 min to conduct with the standard amine coupling procedure. Aiphanol was dissolved in DMSO and diluted in buffer containing 10 mM MOPS (pH 7.2), 150 mM NaCl, 1 mM MgCl 2 and 0.05% Surfactant P20, followed by injection over the functionalized surface at a flow rate of 20 µL/min. The sensorgrams and the kinetic parameters were determined by Biacore S200 evaluation software 2.1.

BioLayer Interferometry (BLI)
The binding between VEGF and VEGFR2 protein were detected using the Octet RED96 System from Forté Bio (Menlo Park, USA). VEGFR2 was conjugated to EZ-Link NHS-PEG12-Biotin and diluted to 10 g/mL by HBS-EP+ buffer (GE Healthcare). Streptavidin sensors were pre-wetted by dipping into HBS-EP+ buffer for 15 min and loaded with biotinylated VEGFR2 for 720 s to establish a baseline signal and diluted with HBS-EP+ buffer for 180 s to remove non-specific adsorption. Then the sensors were immersed into different concentrations of VEGF solution to associate for 300 s and dissociate for 360 s in HBS-EP+ buffer.
Sensorgrams were obtained at each concentration and the Kd was calculated by the FortéBio Data Analysis 12.0. To evaluate the interfering effect of Aiphanol on VEGF-VEGFR2 interaction, 2x Kd concentration of VEGF and different concentrations of Aiphanol were mixed at same volume. Bevacizumab (Bev) was used as the positive control to inhibit VEGF-VEGFR2 association. Then the immobilized sensors were dipped into the mixture for association and returned to HBS-EP+ buffer for dissociation. Finally, data analysis was performed using a reference subtraction and aligned using baseline signal.

ADP-Glo TM assay
The ADP-Glo TM Kinase Kit from Promega (Madison, USA) was used to evaluate Aiphanol's effect on VEGFR2 kinase activity. The kinase buffer mixture contained different concentrations of Aiphanol, 1.5 ng/μL VEGFR2, 0.2 μg/μL poly (4:1 Glu, Tyr) peptide, and 50 μM ATP. The reaction was started by addition of ATP at 37°C for 1 h. 5 µL ADP-Glo TM reagent was added to each well to stop the reaction and incubated at room temperature for 40 min. Finally, 10 µL kinase detection reagent was added to the wells and incubated for 30 min to generate luminescent signals, which were measured under an automated microplate reader and the dose-response curve was fitted.

siRNA interference assay
Small interfering RNAs (siRNA) were synthesized by GenePharma (Suzhou, China were set according to the results of the pre-assay. Drugs were injected into the yolk sac of zebrafish (for) 48 h after fertilization by the microinjection method. After incubation for 24 h at 28°C, fluorescence of the subintestinal vessels (SIVs) was observed and photographed using a multi-purpose zoom microscope system (Nikon, Japan). Image analysis was performed by calculating the SIVs area via Nikon NIS-Elements D 3.10 Advanced image processing software.

Matrigel plug assay
Matrigel plug angiogenesis assay was performed following previously reported protocol with some modifications. 4

Mouse aortic ring assay
The procedures were reported previously. 5 The 48-well plates were coated with 120 μL of Matrigel per well and polymerized at 37°C for 1 h. Aortas harvested from 7-week-old C57BL/6 mice were sliced into 1 mm thick rings. The rings were placed on the polymerized Matrigel and sealed with 60 μL Matrigel for 3 h to allow Matrigel to be polymerized firmly. 500 μL serum-free EBM-2 medium containing VEGF (20 ng/mL) with or without indicated concentrations of Aiphanol was added into the wells. Bevacizumab (Bev, 0.5 mg/mL) was used as the positive control. After 6 d, the microvessel area was photographed and analyzed with Image J.

Chicken embryo chorioallantoic membrane (CAM) assay
CAM assay was performed following previously reported procedures with some modifications. 2 Briefly, fresh fertilized white leghorn chicken eggs from Boehringer Ingelheim Vital Biotechnology (Beijing, China) were swabbed by 1% bromogeramine and incubated at 37°C with 60% humidity. On the third day of embryo development, the whole egg contents were gently transferred into sterilized dishes and incubated at 37°C.
Glass cellulose filters, which were soaked with VEGF (20 ng/mL) with or without indicated concentrations of Aiphanol, were applied to CAMs. Bevacizumab (Bev, 0.5 mg/mL) was used as the positive control. Then the embryos were cultured for 48 h. Vascular response to drugs was evaluated by counting the number of blood vessels within the defined area of the filters under stereomicroscope (Olympus, Japan).

MC38 syngeneic mouse model
C57BL/6 mice were subcutaneously injected with 7 x 10 5 MC38 cells which were resuspended in PBS containing 50% growth factor reduced Matrigel. Aiphanol was dissolved in the solvent (10% DMSO, 40% PEG300, 3% Tween-80, 47% saline). Upon tumors reaching 100 mm 3 , mice were administered orally with 100 L of Aiphanol (30 mg/kg for single-dosage experiment, 5 and 30 mg/kg for double-dosage experiment) or solvent control once daily. Bevacizumab (Bev, 5 mg/kg) was injected through tail vein as the positive control every four days in the double-dosage experiment. Tumor volume was measured thrice weekly and calculated as width 2 x length/2. Mice were sacrificed after 12 d (for single-dosage experiment) or 8 d (for double-dosage experiment), the tumors were resected and weighed.

Immunohistochemistry (IHC)
The tumors from single-dosage experiment were resected and fixed in 10% buffered formaldehyde for 48 h.
Tumors were paraffin-embedded, sectioned (4 µm thickness), deparaffinized and hydrated. Antigen retrieval was performed by autoclave heating in Tris-EDTA buffer (pH 9.0) for 15 min. Sections were then quenched with 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity and blocked with normal goat serum for 40 min at room temperature. Primary antibodies were applied to the sections overnight at 4°C. After probing with secondary antibodies, the peroxidase reaction was carried out with Liquid DAB Substrate (GeneTech, USA). Finally, slides were counterstained with hematoxylin (ZSGB Biotechnology). The intensity of IHC staining was quantified by the IHC Profiler plugin in Image J.

Frozen sections
The resected tumors from single-dosage experiment were flash-frozen in liquid nitrogen and immediately embedded in optimal-cutting-temperature (OCT) compound (Sakura) for sectioning with the freezing microtome (Leica, Germany) by the pathologist. Frozen sections were subjected to immunofluorescence staining analysis of CD31 expression.

Statistical Analysis
Graph Pad Prism 6.0 software was used to perform an ANOVA test and the Student's two-tailed unpaired t-test.